Gene therapy for treating phenylketonuria

ABSTRACT

Compositions and regimens useful in treating phenylketonuria are provided. The compositions include recombinant adeno-associated virus (rAAV) with a transthyretin enhancer and promoter driving expression of a human phenylalanine hydroxylase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of US Patent Application No.16/474,960, filed Jun. 28, 2019, which is a National Stage Entry under35 U.S.C. 371 of International Patent Application No. PCT/US2017/068897,filed Dec. 29, 2017, which claims the benefit of priority of USProvisional Patent Application No. 62/440,651, filed Dec. 30, 2016, USProvisional Patent Application No. 62/469,898, filed Mar. 10, 2017, andUS Provisional Patent Application No. 62/505,373, filed May 12, 2017.These applications are incorporated by reference herein.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeled “UPN-16-7939PCTST25.txt”.

INTRODUCTION

The application relates to embodiments useful for a gene therapy fortreating phenylketonuria.

BACKGROUND

As one of the most common inborn errors of metabolism, Phenylketonuria(PKU) occurs in 1 in 10,000 to 15,000 newborns in the United States. Thecurrent treatment approaches require the affected individual to adhereconsistently to an unpalatable and expensive dietary restriction and/ortake enzyme substitution with phenylalanine ammonia lyase from birth fortheir whole life.

The most common cause of PKU is deficiency of phenylalanine hydroxylase(PAH) due to a recessively inherited mutation in the PAH gene. PAH isexpressed primarily in the liver that catalyzes the irreversiblehydroxylation of phenylalanine to tyrosine. Thus, deficiency in PAHaffects the catabolic pathway of phenylalanine, resulting inaccumulation of phenylalanine. High plasma phenylalanine levels resultsin build-up of phenylalanine in the brain and can affect braindevelopment and function, resulting in intellectual disability andseizures. Furthermore, reduction of plasma phenylalanine via dietaryrestriction and enzyme substitution is expensive, inconvenient and hasbeen linked with various adverse complications, such as persistent mildcognitive deficits.

An alternative approach to achieve sustained therapeutic levels of PAHis through continuous in vivo production of the native enzyme in thehepatocytes using gene transfer mediated by a cell-directedadeno-associated virus (AAV) or other viral or non-viral vector. Severalattempts of vector-mediated PAH expression have been tested preliminaryon mouse studies. See, e.g., Harding et al, Complete correction ofhyperphenylalaninemia following liver-directed, recombinant AAV2/8vector mediated gene therapy in murine phenylketonuria Gene Ther. 2006Mar; 13(5):457-6 and Viecelli et al, Treatment of Phenylketonuria UsingMinicircle-Based Naked-DNA Gene Transfer to Murine Liver Hepatology.2014 September; 60(3): 1035-1043, which are incorporated herein byreference. However, the evaluations of delivery efficiency, immunestimulation, long-term expression stability and safety are eitherlacking or not optimal. Thus, more efficient AAV.hPAH vectors are neededfor PKU treatment.

SUMMARY

The embodiments described herein relate to an AAV gene therapy vectorfor delivering normal human phenylalanine hydroxylase (PAH) to a subjectin need thereof, following intravenous administration of the vectorresulting in long-term, perhaps 10 years or more, of clinicallymeaningful correction of hyperphenylalaninemia. The subject patientpopulation is patients with moderate to severe hyperphenylalaninemia,including those with PKU, variant PKU or non-PKU hyperphenylalaninemia.The intended vector dose is intended to deliver PAH blood levels ofapproximately 15% or greater as compared to wild type, which is thelevel which has been reported for “moderate” PKU patients. See, Kaufman,S., PNAS, 96:3160-4 (1999), which is incorporated herein by reference.In another embodiment, the intended vector dose is intended to deliverPAH to result in a reduction of plasma phenylalanine levels by 25% orgreater. In one embodiment, the goal for the AAV vector treatment isconversion of severe PKU patients to either moderate or mild PKU thuslessening the burden associated with a severely limited phenylalaninediet.

In one aspect, this application provides the use of a replicationdeficient adeno-associated virus (AAV) to deliver a human phenylalaninehydroxylase (PAH) gene to liver cells of patients (human subjects)diagnosed with PKU. The recombinant AAV vector (rAAV) used fordelivering the hPAH gene (“rAAV.hPAH”) should have a tropism for theliver (e.g., a rAAV bearing an AAV8 capsid), and the hPAH transgeneshould be controlled by liver-specific expression control elements. Inone embodiment, the expression control elements include one or more ofthe following: an enhancer; a promoter; an intron; a WPRE; and a polyAsignal. Such elements are further described herein.

In one embodiment, the hPAH coding sequence is shown in SEQ ID NO: 1. Inone embodiment, the PAH protein sequence is shown in SEQ ID NO: 2. Thecoding sequence for hPAH is, in one embodiment, codon optimized forexpression in humans. Such sequence may share less than 80% identity tothe native hPAH coding sequence (SEQ ID NO: 3). In one embodiment, thehPAH coding sequence is that shown in SEQ ID NO: 1.

In another aspect, provided herein is an aqueous suspension suitable foradministration to a PKU patient which includes the rAAV describedherein. In some embodiments, the suspension includes an aqueoussuspending liquid and about 1×10¹² to about 1×10¹⁴ genome copies (GC) ofthe rAAV/mL. The suspension is, in one embodiment, suitable forintravenous injection. In other embodiment, the suspension furtherincludes a surfactant, preservative, and/or buffer dissolved in theaqueous suspending liquid.

In another embodiment, provided herein is a method of treating a patienthaving PKU with an rAAV as described herein. In one embodiment, about1×10¹¹to about 3×10¹³ genome copies (GC) of the rAAV/kg patient bodyweight are delivered the patient in an aqueous suspension.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of pAAV.TBG.PI.hPAHco.WPRE.bGH cisplasmid.

FIG. 2A is a bar graph of plasma phenylalanine (Phe) levels in PAH_KO_Amouse model (shown in white) and wild-type (shown in black) orheterozygous (shown in grey) littermates, as described in Example 1.These results are summarized in FIG. 2D.

FIG. 2B is a line graph of plasma phenylalanine (Phe) levels in PAH_KO_Amice (shown in white) with heterozygous (shown in grey) and wild-type(shown in black) littermates provided as controls. Mice were injectedwith 1×10¹³ GC/kg or 1×10¹² GC/kg of AAV8.TBG.PI.hPAHco.WPRE.bGH on day56 of natural history study as described in Example 3. Experiment wasperformed on 7 male and 3 female PAH_KO_A mice from the natural historystudy.

FIG. 2C is a line graph of plasma phenylalanine (Phe) levels in PAH_KO_Amouse model injected with 1×10¹³ GC/kg or 1×10¹² GC/kg ofAAV8.TBG.PI.hPAHco.WPRE.bGH as described in Example 3. The day ofinjection was Day 0.

FIG. 2D is a line graph showing the mean Phe levels for the mice studiedin FIG. 2A. Values expressed as mean +/−SEM.

FIG. 3A is a bar graph of plasma phenylalanine (Phe) levels in PAH_KO_Bmouse model (shown in white) and wild-type (shown in black) orheterozygous (shown in grey) littermates, as described in Example 1.These results are summarized in FIG. 3D.

FIG. 3B is a line graph of plasma phenylalanine (Phe) levels in PAH_KO_Bmice (shown in white) with heterozygous (shown in grey) and wild-type(shown in black) littermates provided as controls. Experiment wasperformed on 3 female PAH_KO_B mice from the natural history study.

FIG. 3C is a line graph of plasma phenylalanine (Phe) levels in PAH_KO_Bmouse model injected with 1 x 10¹² GC/kg of AAV8.TBG.PI.hPAHco.WPRE.bGHas described in Example 3. Mice Identification Numbers 1691, 1695 and1696 were females injected with 1×10¹² GC/kg on Day 0.

FIG. 3D is a line graph showing the mean Phe levels for the mice studiedin FIG. 3A. Values expressed as mean +/−SEM. FIG. 4A is a line graph ofplasma phenylalanine (Phe) levels in PAH_KO_C mouse model (shown inwhite) and wild-type (shown in black) or heterozygous (shown in grey)littermates, as described in Example 1. These results are summarized inFIG. 4C.

FIG. 4B is a line graph of plasma phenylalanine (Phe) levels in PAH_KO_Cmice (shown in white) with heterozygous (shown in grey) and wild-type(shown in black) littermates provided as controls. Experiment wasperformed on 2 male PAH_KO_C mice from the natural history study.

FIG. 4C is a line graph showing the mean Phe levels for the mice studiedin FIG. 4A. Values expressed as mean +/−SEM.

FIG. 5 is a bar graph summarizing the results of FIGS. 2A, 3A and 4A.Plasma phenylalanine (Phe) levels were detected via LC/MS/MS and thedata from PAH_KO_A, PAH_KO_B and PAH_KO_C mice bled from 6-8 weeks ofage. Plasma was isolated and analyzed for Phe concentration. Wild-typelittermates were provided as negative controls.

FIG. 6A-6C demonstrate that AAV8.TBG.hPAHco rescues phenylalanine levelsin PKU KO B mice. PKU B mice ages 17-22 weeks were given either 10¹²GC/kg of either AAV8.TBG.hPAHco.bGH (circles) orAAV8.TBG.hPAHco.WPRE.bGH (squares) after pretreatment phenylalaninelevels were established. PBS treatment shown with triangles. Mice werethen bled weekly, and plasma was isolated and phenylalanineconcentration was determined (A). At the termination of the study, liverwas collected and genome copy analysis (B) and immunohistochemistry (C)was performed. Values expressed as mean±SEM.

FIG. 7 is a line graph demonstrating that AAV gene therapy lowers plasmaPhe concentration in PKU_KO_A mice. WT (triangle), heterozygous (circle)and PKU_KO_A (KO) mice were injected intravenously with 10¹¹ GC/kg ofAAV8.TBG.hPAHco after baseline phenylalanine levels were established.Mice were then bled weekly, and plasma was isolated and analyzed for Pheconcentration. Values expressed as mean +/−SEM. Phe levels in plasmadecreased by 71% following intravenous administration ofAAV8.TBG.hPAHco.

FIGS. 8A-8C demonstrate that high does AAV9.TBG.hPAHco rescuesphenylalanine levels in PKU_KO_B mice. PKU_KO_B mice were given either10¹² GC/kg, 3×10¹¹ GC/kg, or 10¹¹ GC/kg, of AAV8.TBG.hPAHco afterbaseline phenylalanine levels were established. Mice were then bledweekly, and plasma was isolated and analyzed for Phe concentration (A).Values expressed as mean +/−SEM. At termination of the study, liver wascollected and genome copy analysis (B) and immunohistochemistry (C) wasperformed. Protein expression and reduction in Phe levels seen at a doseof 10¹² GC/kg.

FIG. 9 shows an alignment of a portion of the PAH sequence for wild type(WT), PAH_KO_A (Strain A), PAH_KO_B (Strain B), PAH_KO_C (Strain C),PAH_KO_D (Strain D) and consensus.

DETAILED DESCRIPTION

The embodiments described in the application relate to the use of areplication deficient adeno-associated virus (AAV) to deliver a humanphenylalanine hydroxylase (PAH) gene to liver cells of patients (humansubjects) diagnosed with phenylketonuria (PKU). The recombinant AAVvector (rAAV) used for delivering the hPAH gene (“rAAV.hPAH”) shouldhave a tropism for the liver (e.g., a rAAV bearing an AAV8 capsid), andthe hPAH transgene should be controlled by liver-specific expressioncontrol elements. In one embodiment, the expression control elementsinclude one or more of the following: an enhancer; a promoter; anintron; a WPRE; and a polyA signal. Such elements are further describedherein.

As used herein, “AAV8 capsid” refers to the AAV8 capsid having the aminoacid sequence of GenBank, accession: YP_077180.1, SEQ ID NO: 19, whichis incorporated by reference herein. Some variation from this encodedsequence is permitted, which may include sequences having about 99%identity to the referenced amino acid sequence in YP_077180.1 and WO2003/052051 (which is incorporated herein by reference) (i.e., less thanabout 1% variation from the referenced sequence). Methods of generatingthe capsid, coding sequences therefore, and methods for production ofrAAV viral vectors have been described. See, e.g., Gao, et al, Proc.Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2015/0315612.

As used herein, the term “NAb titer” a measurement of how muchneutralizing antibody (e.g., anti-AAV Nab) is produced which neutralizesthe physiologic effect of its targeted epitope (e.g., an AAV). Anti-AAVNAb titers may be measured as described in, e.g., Calcedo, R., et al.,Worldwide Epidemiology of Neutralizing Antibodies to Adeno-AssociatedViruses. Journal of Infectious Diseases, 2009. 199(3): p. 381-390, whichis incorporated by reference herein.

The terms “percent (%) identity”, “sequence identity”, “percent sequenceidentity”, or “percent identical” in the context of amino acid sequencesrefers to the residues in the two sequences which are the same whenaligned for correspondence. Percent identity may be readily determinedfor amino acid sequences over the full-length of a protein, polypeptide,about 32 amino acids, about 330 amino acids, or a peptide fragmentthereof or the corresponding nucleic acid sequence coding sequencers. Asuitable amino acid fragment may be at least about 8 amino acids inlength, and may be up to about 700 amino acids. Generally, whenreferring to “identity”, “homology”, or “similarity” between twodifferent sequences, “identity”, “homology” or “similarity” isdetermined in reference to “aligned” sequences. “Aligned” sequences or“alignments” refer to multiple nucleic acid sequences or protein (aminoacids) sequences, often containing corrections for missing or additionalbases or amino acids as compared to a reference sequence. Alignments areperformed using any of a variety of publicly or commercially availableMultiple Sequence Alignment Programs. Sequence alignment programs areavailable for amino acid sequences, e.g., the “Clustal X”, “MAP”,“PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs.Generally, any of these programs are used at default settings, althoughone of skill in the art can alter these settings as needed.Alternatively, one of skill in the art can utilize another algorithm orcomputer program which provides at least the level of identity oralignment as that provided by the referenced algorithms and programs.See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensivecomparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

As used herein, the term “operably linked” refers to both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest.

A “replication-defective virus” or “viral vector” refers to a syntheticor artificial viral particle in which an expression cassette containinga gene of interest is packaged in a viral capsid or envelope, where anyviral genomic sequences also packaged within the viral capsid orenvelope are replication-deficient; i.e., they cannot generate progenyvirions but retain the ability to infect target cells. In oneembodiment, the genome of the viral vector does not include genesencoding the enzymes required to replicate (the genome can be engineeredto be “gutless” - containing only the transgene of interest flanked bythe signals required for amplification and packaging of the artificialgenome), but these genes may be supplied during production. Therefore,it is deemed safe for use in gene therapy since replication andinfection by progeny virions cannot occur except in the presence of theviral enzyme required for replication.

It is to be noted that the term “a” or “an” refers to one or more. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” areused interchangeably herein.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively. The words “consist”,“consisting”, and its variants, are to be interpreted exclusively,rather than inclusively. While various embodiments in the specificationare presented using “comprising” language, under other circumstances, arelated embodiment is also intended to be interpreted and describedusing “consisting of” or “consisting essentially of” language.

As used herein, the term “about” means a variability of 10% from thereference given, unless otherwise specified.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art and by reference to published texts, whichprovide one skilled in the art with a general guide to many of the termsused in the present application.

5.1 Gene Therapy Vectors

In one aspect, a recombinant adeno-associated virus (rAAV) vectorcarrying the human PAH gene is provided for use in gene therapy. TherAAV.hPAH vector should have a tropism for the liver (e.g., a rAAVbearing an AAV8 capsid) and the hPAH transgene should be controlled byliver-specific expression control elements. The vector is formulated ina buffer/carrier suitable for infusion in human subjects. Thebuffer/carrier should include a component that prevents the rAAV fromsticking to the infusion tubing but does not interfere with the rAAVbinding activity in vivo.

5.1.1. The rAAV.hPAH Vector 5.1.1.1. The hPAH Sequence

Phenylketonuria is an inherited error of metabolism caused predominantlyby mutations in the phenylalanine hydroxylase (PAH) gene. Mutations inthe PAH gene result in decreased catalytic activity affecting thecatabolic pathway of phenylalanine (Phe). PAH is a hepatic enzyme thatrequires the cofactor tetrahydrobiopterin (BH4) to convert Phe totyrosine (Tyr). A deficiency in PAH or its cofactor BH4 results in theaccumulation of excess phenylalanine, whose toxic effects can causesevere and irreversible intellectual disability and other disorders, ifuntreated. See, Havid and Cristodoulou, Transl Pediatr, 2015 October,4(4):304-17, which is incorporated herein by reference.

Over 550 mutations of the PAH gene have been described, the majority ofwhich result in deficient enzyme activity. See, PhenylalanineHydroxylase Locus Knowledgebase, accessed online at pandb.mcgill.ca/,which is incorporated herein by reference. Due to the large number ofknown PKU mutations, and the autosomal recessive nature of the disease,a wide range of disease severity is seen. The severity of the disease isgenerally classified by blood phenylalanine levels, which are sometimesclassified as classic PKU, moderate or variant PKU, mild PKU, orhyperphenylalaninemia. Based on blood Phe levels at diagnosis, there are4 levels of PKU severity.

-   -   Hyperphenylalaninemia, with Phe levels that are slightly above        normal range: 120-600 μmol/L (2-10 mg/dL)    -   Mild, with the lowest blood Phe levels: 600-900 μmol/L (10-15        mg/dL)    -   Moderate or variant, with blood Phe levels somewhere in the        middle: 900-1200 μmol/L (15-20 mg/dL)    -   Severe or “classic” PKU, with extremely high blood Phe        levels: >1200 μmol/L (20 mg/dL)

The goal of therapies described herein would provide functional PAHenzyme resulting in Phe levels in the 120-600 μmol/L range, e.g., a 25%or greater reduction in plasma Phe levels. In another embodiment, thevector dose is intended to deliver PAH to result in a reduction ofplasma phenylalanine levels by 25% or greater.

In another embodiment, the vector dose is intended to deliver PAH toresult in a reduction of plasma phenylalanine levels by 30% or greater.In another embodiment, the vector dose is intended to deliver PAH toresult in a reduction of plasma phenylalanine levels by 35% or greater.In another embodiment, the vector dose is intended to deliver PAH toresult in a reduction of plasma phenylalanine levels by 40% or greater.In another embodiment, the vector dose is intended to deliver PAH toresult in a reduction of plasma phenylalanine levels by 45% or greater.In another embodiment, the vector dose is intended to deliver PAH toresult in a reduction of plasma phenylalanine levels by 50% or greater.In another embodiment, the vector dose is intended to deliver PAH toresult in a reduction of plasma phenylalanine levels by 60% or greater.In another embodiment, the vector dose is intended to deliver PAH toresult in a reduction of plasma phenylalanine levels by 70% or greater.In another embodiment, the vector dose is intended to deliver PAH toresult in a reduction of plasma phenylalanine levels by 75% or greater.

In one embodiment, the “subject” or “patient” is a mammalian subjecthaving PKU as described above. It is intended that a patient having PKUof any severity is the intended subject.

In one embodiment, the hPAH gene encodes the hPAH protein shown in SEQID NO: 2. Thus, in one embodiment, the hPAH transgene can include, butis not limited to, the sequence provided by SEQ ID NO:1 or SEQ ID NO: 3which are provided in the attached Sequence Listing, which isincorporated by reference herein. SEQ ID NO: 3 provides the cDNA fornative human PAH. SEQ ID NO: 1 provides an engineered cDNA for humanPAH, which has been codon optimized for expression in humans (sometimesreferred to herein as hPAHco). It is to be understood that reference tohPAH herein may, in some embodiments, refer to the hPAH native or codonoptimized sequence. Alternatively or additionally, web-based orcommercially available computer programs, as well as service basedcompanies may be used to back translate the amino acid sequences tonucleic acid coding sequences, including both RNA and/or cDNA. See,e.g., backtranseq by EMBOSS, http://www.ebi.ac.uk/Tools/st/; GeneInfinity (http://www.geneinfinity.org/sms-/sms_backtranslation.html);ExPasy (http://www.expasy.org/tools/). It is intended that all nucleicacids encoding the described hPAH polypeptide sequences are encompassed,including nucleic acid sequences which have been optimized forexpression in the desired target subject (e.g., by codon optimization).

In one embodiment, the nucleic acid sequence encoding hPAH shares atleast 95% identity with the native hPAH coding sequence of SEQ ID NO: 3.In another embodiment, the nucleic acid sequence encoding hPAH shares atleast 90, 85, 80, 75, 70, or 65% identity with the native hPAH codingsequence of SEQ ID NO: 3. In one embodiment, the nucleic acid sequenceencoding hPAH shares about 78% identity with the native hPAH codingsequence of SEQ ID NO: 3. In one embodiment, the nucleic acid sequenceencoding hPAH is SEQ ID NO: 1.

In one embodiment, the PAH coding sequence is optimized for expressionin the target subject. Codon-optimized coding regions can be designed byvarious different methods. This optimization may be performed usingmethods which are available on-line (e.g., GeneArt,), published methods,or a company which provides codon optimizing services, e.g., as DNA2.0(Menlo Park, Calif.). One codon optimizing approach is described, e.g.,in International Patent Publication No. WO 2015/012924, which isincorporated by reference herein. See also, e.g., US Patent PublicationNo. 2014/0032186 and US Patent Publication No. 2006/0136184. Suitably,the entire length of the open reading frame (ORF) for the product ismodified. However, in some embodiments, only a fragment of the ORF maybe altered. By using one of these methods, one can apply the frequenciesto any given polypeptide sequence, and produce a nucleic acid fragmentof a codon-optimized coding region which encodes the polypeptide.

A number of options are available for performing the actual changes tothe codons or for synthesizing the codon-optimized coding regionsdesigned as described herein. Such modifications or synthesis can beperformed using standard and routine molecular biological manipulationswell known to those of ordinary skill in the art. In one approach, aseries of complementary oligonucleotide pairs of 80-90 nucleotides eachin length and spanning the length of the desired sequence aresynthesized by standard methods. These oligonucleotide pairs aresynthesized such that upon annealing, they form double strandedfragments of 80-90 base pairs, containing cohesive ends, e.g., eacholigonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8,9, 10, or more bases beyond the region that is complementary to theother oligonucleotide in the pair. The single-stranded ends of each pairof oligonucleotides are designed to anneal with the single-stranded endof another pair of oligonucleotides. The oligonucleotide pairs areallowed to anneal, and approximately five to six of thesedouble-stranded fragments are then allowed to anneal together via thecohesive single stranded ends, and then they ligated together and clonedinto a standard bacterial cloning vector, for example, a TOPO® vectoravailable from Thermo Fisher Scientific Inc. The construct is thensequenced by standard methods. Several of these constructs consisting of5 to 6 fragments of 80 to 90 base pair fragments ligated together, i.e.,fragments of about 500 base pairs, are prepared, such that the entiredesired sequence is represented in a series of plasmid constructs. Theinserts of these plasmids are then cut with appropriate restrictionenzymes and ligated together to form the final construct. The finalconstruct is then cloned into a standard bacterial cloning vector, andsequenced. Additional methods would be immediately apparent to theskilled artisan. In addition, gene synthesis is readily availablecommercially.

5.1.1.2. The rAAV Vector

Because PAH is natively expressed in the liver, it is desirable to usean AAV which shows tropism for liver. In one embodiment, the AAVsupplying the capsid is AAV8. In another embodiment, the AAV supplyingthe capsid is AAVrh.10. In yet another embodiment, the AAV supplying thecapsid is a Clade E AAV. Such AAV include rh.2; rh.10; rh. 25; bb.1,bb.2, pi.1, pi.2, pi.3, rh.38, rh.40, rh.43, rh.49, rh.50, rh.51, rh.52,rh.53, rh.57, rh.58, rh.61, rh.64, hu.6, hu.17, hu.37, hu.39, hu.40,hu.41, hu.42, hu.66, and hu.67. This clade further includes modified rh.2; modified rh. 58; and modified rh.64. See, WO 2005/033321, which isincorporated herein by reference. However, any of a number of rAAVvectors with liver tropism can be used.

In a specific embodiment described in the Examples, infra, the genetherapy vector is an AAV8 vector expressing an hPAH transgene undercontrol of a thyroxine binding globulin (TBG) promoter referred to asAAV8.TBG.PI.hPAHco.WPRE.bGH. The vector genome for such a vector isshown in SEQ ID NO: 20. In another embodiment, the WPRE is omitted,i.e., AAV8.TBG.PI.hPAHco.bGH. The vector genome for such a vector isshown in SEQ ID NO: 21. The external AAV vector component is a serotype8, T=1 icosahedral capsid consisting of 60 copies of three AAV viralproteins, VP1, VP2, and VP3, at a ratio of 1:1:10. The capsid contains asingle-stranded DNA rAAV vector genome.

In one embodiment, the rAAV.hPAH genome contains an hPAH transgeneflanked by two AAV inverted terminal repeats (ITRs). In one embodiment,the hPAH transgene includes one or more of an enhancer, promoter, anintron, a Woodchuck Hepatitis Virus (WHP) Posttranscriptional RegulatoryElement (WPRE) (e.g., SEQ ID NO: 15), an hPAH coding sequence, andpolyadenylation (polyA) signal. In another embodiment, the hPAHtransgene includes one or more of an enhancer, promoter, an intron, anhPAH coding sequence, and polyadenylation (polyA) signal. These controlsequences are “operably linked” to the hPAH gene sequences. Theexpression cassette containing these sequences may be engineered onto aplasmid which is used for production of a viral vector.

The ITRs are the genetic elements responsible for the replication andpackaging of the genome during vector production and are the only viralcis elements required to generate rAAV. The minimal sequences requiredto package the expression cassette into an AAV viral particle are theAAV 5′ and 3′ ITRs, which may be of the same AAV origin as the capsid,or which of a different AAV origin (to produce an AAV pseudotype). Inone embodiment, the ITR sequences from AAV2, or the deleted versionthereof (ΔITR), are used. However, ITRs from other AAV sources may beselected. Where the source of the ITRs is from AAV2 and the AAV capsidis from another AAV source, the resulting vector may be termedpseudotyped. Typically, an expression cassette for an AAV vectorcomprises an AAV 5′ ITR, the hPAH coding sequences and any regulatorysequences, and an AAV 3′ ITR. However, other configurations of theseelements may be suitable. A shortened version of the 5′ ITR, termedΔITR, has been described in which the D-sequence and terminal resolutionsite (trs) are deleted. In other embodiments, the full-length AAV 5′ and3′ ITRs are used. In one embodiment, the 5′ ITR is that shown in SEQ IDNO: 16. In one embodiment, the 3′ ITR is that shown in SEQ ID NO: 17.

In one embodiment, the expression control sequences include one or moreenhancer. In one embodiment, the En34 enhancer is included (34 bp coreenhancer from the human apolipoprotein hepatic control region), which isshown in SEQ ID NO: 4. In another embodiment, the EnTTR (100 bp enhancersequence from transthyretin) is included. Such sequence is shown in SEQID NO: 5. See, Wu et al, Molecular Therapy, 16(2):280-289, February2008, which is incorporated herein by reference. In yet anotherembodiment, the α1-microglogulin/bikunin precursor enhancer is included.In yet another embodiment, the ABPS (shortened version of the 100 bpdistal enhancer from the al-microglogulin/bikunin precursor [ABP] to 42bp) enhancer is included. Such sequence is shown in SEQ ID NO: 6. In yetanother embodiment, the ApoE enhancer is included. Such sequence isshown in SEQ ID NO: 7. In another embodiment, more than one enhancer ispresent. Such combination may include more than one copy of any of theenhancers described herein, and/or more than one type of enhancer.

Expression of the hPAH coding sequence is driven from a liver-specificpromoter. An illustrative plasmid and vector described herein uses thethyroxine binding globulin (TBG) promoter (SEQ ID NO: 9), or a modifiedversion thereof. One modified version of the TBG promoter is a shortenedversion, termed TBG-S1. A modified thyroxine binding globulin (TBG-S1)promoter sequence is shown in SEQ ID NO: 8. Alternatively, otherliver-specific promoters may be used such as the transthyretin promoter.Another suitable promoter is the alpha 1 anti-trypsin (A1AT) promoter,or a modified version thereof (which sequence is shown in SEQ ID NO:10). Another suitable promoter is the TTR promoter (SEQ ID NO: 11).Other suitable promoters include human albumin (Miyatake et al., J.Virol., 71:5124 32 (1997)), humAlb; the Liver Specific promoter (LSP),and hepatitis B virus core promoter, (Sandig et al., Gene Ther., 3:10029 (1996). See, e.g., The Liver Specific Gene Promoter Database, Cold

Spring Harbor, available online at rulai.schl.edu/LSPD, which isincorporated by reference. Although less desired, other promoters, suchas viral promoters, constitutive promoters, regulatable promoters [see,e.g., WO 2011/126808 and WO 2013/04943], or a promoter responsive tophysiologic cues may be used may be utilized in the vectors describedherein.

In addition to a promoter, an expression cassette and/or a vector maycontain other appropriate transcription initiation, termination,enhancer sequences, and efficient RNA processing signals. Such sequencesinclude splicing and polyadenylation (polyA) signals; regulatoryelements that enhance expression (i.e., WPRE (SEQ ID NO: 15)); sequencesthat stabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance secretion ofthe encoded product. In one embodiment, a polyadenylation (polyA) signalis included to mediate termination of hPAH mRNA transcripts. Examples ofother suitable polyA sequences include, e.g., bovine growth hormone (SEQID NO: 12), SV40, rabbit beta globin, and TK polyA, amongst others.

In one embodiment, the regulatory sequences are selected such that thetotal rAAV vector genome is about 2.0 to about 5.5 kilobases in size. Inone embodiment, the regulatory sequences are selected such that thetotal rAAV vector genome is about 3.4 kb, about 2.9 kb, about 3.3 kb,about 2.2 kb or about 2.5 kb in size. In one embodiment, it is desirablethat the rAAV vector genome approximate the size of the native AAVgenome. Thus, in one embodiment, the regulatory sequences are selectedsuch that the total rAAV vector genome is about 4.7 kb in size. Inanother embodiment, the total rAAV vector genome is less about 5.2 kb insize. The size of the vector genome may be manipulated based on the sizeof the regulatory sequences including the promoter, enhancer, intron,poly A, etc. See, Wu et al, Mol Ther, January 2010 18(1):80-6, which isincorporated herein by reference.

Thus, in one embodiment, an intron is included in the vector. Suitableintrons include the human beta globin IVS2 (SEQ ID NO: 13). See, Kellyet al, Nucleic Acids Research, 43(9):4721-32 (2015), which isincorporated herein by reference. Another suitable promoter includes thePromega chimeric intron (SEQ ID NO: 14), sometimes referred to as “PI”).See, Almond, B. and Schenborn, E. T. A Comparison of pCI-neo Vector andpcDNA4/HisMax Vector. [Internet] 2000, which is incorporated herein byreference. Available from:www.promega.com/resources/pubhub/enotes/a-comparison-of-pcineo-vector-and-pcdna4hismax-vector/).Another suitable intron includes the hFIX intron (SEQ ID NO: 18).Various introns suitable herein are known in the art and include,without limitation, those found athttp://bpg.utoledo.edu/˜afedorov/lab/eid.html, which is incorporatedherein by reference. See also, Shepelev V., Fedorov A. Advances in theExon-Intron Database. Briefings in Bioinformatics 2006, 7: 178-185,which is incorporated herein by reference.

In one embodiment, the rAAV vector genome comprises SEQ ID NO: 20, SEQID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO:25.

5.1.2. Compositions

In one embodiment, the rAAV.hPAH virus is provided in a pharmaceuticalcomposition which comprises an aqueous carrier, excipient, diluent orbuffer. In one embodiment, the buffer is PBS. In a specific embodiment,the rAAV.hPAH formulation is a suspension containing an effective amountof rAAV.hPAH vector suspended in an aqueous solution containing 0.001%Pluronic F-68 in TMN200 (200 mM sodium chloride, 1 mM magnesiumchloride, 20 mM Tris, pH 8.0). However, various suitable solutions areknown including those which include one or more of: buffering saline, asurfactant, and a physiologically compatible salt or mixture of saltsadjusted to an ionic strength equivalent to about 100 mM sodium chloride(NaCl) to about 250 mM sodium chloride, or a physiologically compatiblesalt adjusted to an equivalent ionic concentration.

For example, a suspension as provided herein may contain both NaCl andKC1. The pH may be in the range of 6.5 to 8.5, or 7 to 8.5, or 7.5 to 8.A suitable surfactant, or combination of surfactants, may be selectedfrom among Poloxamers, i.e., nonionic triblock copolymers composed of acentral hydrophobic chain of polyoxypropylene (poly(propylene oxide))flanked by two hydrophilic chains of polyoxyethylene (poly(ethyleneoxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxycapryllic glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylenesorbitan fatty acid esters), ethanol and polyethylene glycol. In oneembodiment, the formulation contains a poloxamer. These copolymers arecommonly named with the letter “P” (for poloxamer) followed by threedigits: the first two digits×100 give the approximate molecular mass ofthe polyoxypropylene core, and the last digit×10 gives the percentagepolyoxyethylene content. In one embodiment Poloxamer 188 is selected.The surfactant may be present in an amount up to about 0.0005% to about0.001% of the suspension. In another embodiment, the vector is suspendedin an aqueous solution containing 180 mM sodium chloride, 10 mM sodiumphosphate, 0.001% Poloxamer 188, pH 7.3.

In one embodiment, the formulation is suitable for use in human subjectsand is administered intravenously. In one embodiment, the formulation isdelivered via a peripheral vein by bolus injection. In one embodiment,the formulation is delivered via a peripheral vein by infusion overabout 10 minutes (±5 minutes). In one embodiment, the formulation isdelivered via a peripheral vein by infusion over about 20 minutes (±5minutes). In one embodiment, the formulation is delivered via aperipheral vein by infusion over about 30 minutes (±5 minutes). In oneembodiment, the formulation is delivered via a peripheral vein byinfusion over about 60 minutes (±5 minutes). In one embodiment, theformulation is delivered via a peripheral vein by infusion over about 90minutes (±10 minutes). However, this time may be adjusted as needed ordesired. Any suitable method or route can be used to administer anAAV-containing composition as described herein, and optionally, toco-administer other active drugs or therapies in conjunction with theAAV-mediated delivery of hPAH described herein. Routes of administrationinclude, for example, systemic, oral, inhalation, intranasal,intratracheal, intraarterial, intraocular, intravenous, intramuscular,subcutaneous, intradermal, and other parental routes of administration.

In one embodiment, the formulation may contain, e.g., about 1.0×10¹¹genome copies per kilogram of patient body weight (GC/kg) to about1×10¹⁵ GC/kg, about 5×10¹¹ genome copies per kilogram of patient bodyweight (GC/kg) to about 3×10¹³ GC/kg, or about 1×10¹² to about 1×10¹⁴GC/kg, as measured by oqPCR or digital droplet PCR (ddPCR) as describedin, e.g., M. Lock et al, Hum Gene Ther Methods. 2014 April;25(2):115-25. doi: 10.1089/hgtb.2013.131. Epub 2014 Feb 14, which isincorporated herein by reference. In one embodiment, the rAAV.hPAHformulation is a suspension containing at least 1×10¹³ genome copies(GC)/mL, or greater, as measured by oqPCR or digital droplet PCR (ddPCR)as described in, e.g., M. Lock et al, supra.

In order to ensure that empty capsids are removed from the dose ofAAV.hPAH that is administered to patients, empty capsids are separatedfrom vector particles during the vector purification process, e.g.,using the method discussed herein. In one embodiment, the vectorparticles containing packaged genomes are purified from empty capsidsusing the process described in U.S. Patent Appin No. 62/322,098, filedon Apr. 13, 2016, and entitled “Scalable Purification Method for AAV8”,which is incorporated by reference herein. Briefly, a two-steppurification scheme is described which selectively captures and isolatesthe genome-containing rAAV vector particles from the clarified,concentrated supernatant of a rAAV production cell culture. The processutilizes an affinity capture method performed at a high saltconcentration followed by an anion exchange resin method performed athigh pH to provide rAAV vector particles which are substantially free ofrAAV intermediates. Similar purification methods can be used for vectorshaving other capsids.

While any conventional manufacturing process can be utilized, theprocess described herein (and in U.S. Patent Appin No. 62/322,098)yields vector preparations wherein between 50 and 70% of the particleshave a vector genome, i.e., 50 to 70% full particles. Thus for anexemplary dose of 1.6×10¹² GC/kg, and the total particle dose will bebetween 2.3×10¹² and 3×10¹² particles. In another embodiment, theproposed dose is one half log higher, or 5×10¹² GC/kg, and the totalparticle dose will be between 7.6×10¹² and 1.1×10¹³ particles. In oneembodiment, the formulation is be characterized by an rAAV stock havinga ratio of “empty” to “full” of 1 or less, preferably less than 0.75,more preferably, 0.5, preferably less than 0.3.

A stock or preparation of rAAV8 particles (packaged genomes) is“substantially free” of AAV empty capsids (and other intermediates) whenthe rAAV8 particles in the stock are at least about 75% to about 100%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or at least 99% of the rAAV8 in the stock and “empty capsids”are less than about 1%, less than about 5%, less than about 10%, lessthan about 15% of the rAAV8 in the stock or preparation.

Generally, methods for assaying for empty capsids and AAV vectorparticles with packaged genomes have been known in the art. See, e.g.,Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec.Ther. (2003) 7:122-128. To test for denatured capsid, the methodsinclude subjecting the treated AAV stock to SDS-polyacrylamide gelelectrophoresis, consisting of any gel capable of separating the threecapsid proteins, for example, a gradient gel containing 3-8%Tris-acetate in the buffer, then running the gel until sample materialis separated, and blotting the gel onto nylon or nitrocellulosemembranes, preferably nylon. Anti-AAV capsid antibodies are then used asthe primary antibodies that bind to denatured capsid proteins,preferably an anti-AAV capsid monoclonal antibody, most preferably theB1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000)74:9281-9293). A secondary antibody is then used, one that binds to theprimary antibody and contains a means for detecting binding with theprimary antibody, more preferably an anti-IgG antibody containing adetection molecule covalently bound to it, most preferably a sheepanti-mouse IgG antibody covalently linked to horseradish peroxidase. Amethod for detecting binding is used to semi-quantitatively determinebinding between the primary and secondary antibodies, preferably adetection method capable of detecting radioactive isotope emissions,electromagnetic radiation, or colorimetric changes, most preferably achemiluminescence detection kit. For example, for SDS-PAGE, samples fromcolumn fractions can be taken and heated in SDS-PAGE loading buffercontaining reducing agent (e.g., DTT), and capsid proteins were resolvedon pre-cast gradient polyacylamide gels (e.g., Novex). Silver stainingmay be performed using SilverXpress (Invitrogen, Calif.) according tothe manufacturer's instructions. In one embodiment, the concentration ofAAV vector genomes (vg) in column fractions can be measured byquantitative real time PCR (Q-PCR). Samples are diluted and digestedwith DNase I (or another suitable nuclease) to remove exogenous DNA.After inactivation of the nuclease, the samples are further diluted andamplified using primers and a TaqMan™ fluorogenic probe specific for theDNA sequence between the primers. The number of cycles required to reacha defined level of fluorescence (threshold cycle, CO is measured foreach sample on an Applied. Biosystems Prism 7700 Sequence DetectionSystem. Plasmid DNA containing identical sequences to that contained inthe AAV vector is employed to generate a standard curve in the Q-PCRreaction. The cycle threshold (Ct) values obtained from the samples areused to determine vector genome titer by normalizing it to the Ct valueof the plasmid. standard curve. End-point assays based on the digitalPCR can also be used.

In one aspect, an optimized q-PCR method is provided herein whichutilizes a broad spectrum serine protease, e.g., proteinase K (such asis commercially available from Qiagen). More particularly, the optimizedqPCR genome titer assay is similar to a standard assay, except thatafter the DNase I digestion, samples are diluted with proteinase Kbuffer and treated with proteinase K followed by heat inactivation.Suitably samples are diluted with proteinase K buffer in an amount equalto the sample size. The proteinase K buffer may be concentrated to 2fold or higher. Typically, proteinase K treatment is about 0.2 mg/mL,but may be varied from 0.1 mg/mL to about 1 mg/mL. The treatment step isgenerally conducted at about 55° C. for about 15 minutes, but may beperformed at a lower temperature (e.g., about 37° C. to about 50° C.)over a longer time period (e.g., about 20 minutes to about 30 minutes),or a higher temperature (e.g., up to about 60° C.) for a shorter timeperiod (e.g., about 5 to 10 minutes). Similarly, heat inactivation isgenerally at about 95° C. for about 15 minutes, but the temperature maybe lowered (e.g., about 70 to about 90° C.) and the time extended (e.g.,about 20 minutes to about 30 minutes). Samples are then diluted (e.g.,1000 fold) and subjected to TaqMan analysis as described in the standardassay.

Additionally, or alternatively, droplet digital PCR (ddPCR) may be used.For example, methods for determining single-stranded andself-complementary AAV vector genome titers by ddPCR have beendescribed. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum GeneTher Methods. 2014 April; 25(2):115-25. doi: 10.1089/hgtb.2013.131. Epub2014 Feb. 14.

5.2 Patient Population

As discussed above, a subject having PKU of any severity is the intendedrecipient of the compositions and methods described herein.

Subjects may be permitted to continue their standard of caretreatment(s) (e.g., diet low in Phe; treatment with sapropterindihydrochloride) prior to and concurrently with the gene therapytreatment at the discretion of their caring physician. In thealternative, the physician may prefer to stop standard of care therapiesprior to administering the gene therapy treatment and, optionally,resume standard of care treatments as a co-therapy after administrationof the gene therapy.

Desirable endpoints of the gene therapy regimen are an increase in PAHactivity resulting in Phe levels between 120-360 μmol/L. In anotherembodiment, the vector dose is intended to deliver PAH to result in areduction of plasma phenylalanine levels by 25% or greater. In anotherembodiment, the desirable endpoint is reducing plasma Phe levels to takesubject to a “moderate” phenotype from a “severe” phenotype. Methods formeasurement of phenylalanine levels are known in the art e.g, asdescribed in Gregory et al, Blood phenylalanine monitoring for dietarycompliance among patients with phenylketonuria: comparison of methods,Genetics in Medicine (November 2007) 9, 761-765, which is incorporatedherein by reference. In one embodiment, patients achieve desiredcirculating PAH levels after treatment with rAAV.hPAH, alone and/orcombined with the use of adjunctive treatments.

5.3. Dosing & Route of Administration

In one embodiment, the rAAV.hPAH vector is delivered as a single doseper patient. In one embodiment, the subject is delivered the minimaleffective dose (MED) (as determined by preclinical study described inthe Examples herein). As used herein, MED refers to the rAAV.hPAH doserequired to achieve PAH activity resulting in Phe levels between 120-360μmol/L.

As is conventional, the vector titer is determined on the basis of theDNA content of the vector preparation. In one embodiment, quantitativePCR or optimized quantitative PCR as described in the Examples is usedto determine the DNA content of the rAAV.hPAH vector preparations. Inone embodiment, digital droplet PCR as described in the Examples is usedto determine the DNA content of the rAAV.hPAH vector preparations. Inone embodiment, the dosage is about 1×10¹¹ genome copies (GC)/kg bodyweight to about 1×10¹³ GC/kg, inclusive of endpoints. In one embodiment,the dosage is 5×10¹¹ GC/kg. In another embodiment, the dosage is 5×10¹²GC/kg. In specific embodiments, the dose of rAAV.hPAH administered to apatient is at least 5×10¹¹ GC/kg, 1×10¹² GC/kg, 1.5×10¹² GC/kg, 2.0×10¹²GC/kg, 2.5×10¹² GC/kg, 3.0×10¹² GC/kg, 3.5×10¹² GC/kg, 4.0×10¹² GC/kg,4.5×10¹² GC/kg, 5.0×10¹² GC/kg, 5.5×10¹² GC/kg, 6.0×10¹² GC/kg, 6.5×10¹²GC/kg, 7.0×10¹² GC/kg, or 7.5×10¹² GC/kg. Also, thereplication-defective virus compositions can be formulated in dosageunits to contain an amount of replication-defective virus that is in therange of about 1.0×10⁹ GC to about 1.0×10¹⁵ GC. As used herein, the term“dosage” can refer to the total dosage delivered to the subject in thecourse of treatment, or the amount delivered in a single (of multiple)administration.

In one embodiment, the dosage is sufficient to decrease plasma Phelevels in the patient by 25% or more.

In some embodiments, rAAV.hPAH is administered in combination with oneor more therapies for the treatment of PKU, such as a low Phe diet oradministration of sapropterin dihydrochloride.

5.4. Measuring Clinical Objectives

Measurements of efficacy of treatment can be measured by transgeneexpression and activity as determined by plasma Phe levels and/or PAHactivity. Further assessment of efficacy can be determined by clinicalassessment of dietary Phe tolerance.

As used herein, the rAAV.hPAH vector herein “functionally replaces” or“functionally supplements” the patients defective PAH with active PAHwhen the patient expresses a sufficient level of PAH to achieve PAHactivity resulting in Phe levels between 120-360 μmol/L.

The following examples are illustrative only and are not intended tolimit the present invention.

EXAMPLES

The following examples are illustrative only and are not intended tolimit the present invention.

EXAMPLE 1: Mouse Models of Phenylketonuria (PKU)

PAH^(−/−) mice were generated by CRISPR/Cas9 technology at Jackson Labs.Wild-type C57BL/6 mice were injected with Cas9 mRNA and two guide RNAs(sgRNA) targeting the second coding exon of PAH gene directly into mousezygotes. Mice that developed from these embryos were sequenced todetermine the mutation(s) and then bred with C57BL/6J mice to transmitthe allele and confirm germline transmission.

Four different mutations were generated and the mice strains weredesignated as PAH_KO_A, PAH_KO_B, PAH_KO_C, and PAH_KO_D. PAH_KO_A micedemonstrated a 3-bp substitution followed by a 64-bp deletion from the6534 nt to the 6600 nt of Mus musculus PAH gene [NC_000076.6]. PAH_KO_Band PAH_KO_C mice showed a single base pair insertion after the 6589 ntand the 6539 nt respectively. PAH_KO_D showed a 6 bp deletion from the6535 nt to the 6540 nt. FIG. 9 shows an alignment of a portion of thePAH sequence for wild type, PAH_KO_A, PAH_KO_B, PAH_KO_C, PAH_KO_D andconsensus. A natural history study of these mice was performed. Bloodsamples were collected via retro orbital or submandibular bleeding.Plasma phenylalanine (Phe) levels were detected via LC/MS/MS and thedata from PAH_KO_A, PAH_KO_B and PAH_KO_C mice was acquired andpresented in FIGS. 2A, 3A and 4A respectively, and summarized in FIG.5A. Heterozygous and wild-type littermates were provided as negativecontrols. Compared to the controls, the plasma phenylalanine levels inPAH_KO_A, PAH_KO_B and PAH_KO_C mice were significantly higher,indicating a functionally deficient PAH in these mice. It also suggestedthat PAH_KO_A, PAH_KO_B, and PAH_KO_C mice could serve as mouse modelsfor Phenylketonuria (PKU) in human.

However, the fourth knock-out mice, PAH_KO_D, did not exhibit anelevated plasma phenylalanine level and thus excluded from furtheranalysis.

EXAMPLE 2: AAV Vectors Containing hPAH-AAV8.TBG.PI.hPAHco.WPRE.bGH

The gene therapy vector AAV8.TBG.PI.hPAHco.WPRE.bGH was constructed byan AAV8 vector bearing a codon-optimized human PAH cDNA under thecontrol of TBG, a hybrid promoter based on the human thyroidhormone-binding globulin promoter and microglobin/bikunin enhancer (FIG.1). The PAH expression cassette was flanked by

AAV2 derived inverted terminal repeats (ITRs) and the expression wasdriven by a hybrid of the TBG enhancer/promoter and the WoodchuckHepatitis Virus (WHP) posttranscriptional regulatory element (WPRE) asan enhancer. The transgene also included the Promega SV40 misc intron(PI) and a bovine growth hormone polyadenylation signal (bGH). Thevector genome sequence is shown in SEQ ID NO: 20.

The vector was prepared using conventional triple transfectiontechniques in 293 cells as described e.g., by Mizukami, Hiroaki, et al.A Protocol for AAV vector production and purification. Diss. Di-visionof Genetic Therapeutics, Center for MolecularMedicine, 1998., which isincorporated herein by reference. All vectors were produced by theVector Core at the University of Pennsylvania as previously described[Lock, M., et al, Hum Gene Ther, 21: 1259-1271 (2010)].

EXAMPLE 3: AAV8.hPAH Vectors in the Model of PKU

All animal procedures were performed in accordance with protocolsapproved by the Institutional Animal Care and Use Committee (IACUC) ofthe University of Pennsylvania.

Twenty PAH_KO_A mice were generated. Seven males and three females wereassessed in natural history study. On Day 56 of natural history study,three male mice with Identification Number 1531, 1532 and 1533 wereinjected intravenously via the tail vein with 1×10¹³ GC/kg of theAAV8.TBG.PI.hPAHco.WPRE.bGH vector. Four male mice with IdentificationNumber 1538, 1539, 1554 and 1564 were injected with 1×10¹² GC/kg of thevector. Three female mice with Identification Number 1507, 1536 and 1537were injected with 1×10¹² GC/kg of the vector. The blood samples werecollected weekly to evaluate plasma phenylalanine concentration (FIG.2B). A higher level of phenylalanine was detected in the PAH_KO_A micebefore injection compared to the littermate controls, indicating adeficient PAH in PAH_KO_A mice. 7 days after the vector injection, theplasma phenylalanine level of PAH_KO_A mice decreased while the controlsremained stable. This result demonstrated that a single injection ofAAV8.TBG.PI.hPAHco.WPRE.bGH into PAH_KO_A mice could rescue thedeficient PAH and reduce the pathological accumulation of phenylalaninein the blood.

The effects of gender differences and the two doses of theAAV8.TBG.PI.hPAHco.WPRE.bGH vectors were further evaluated in PAH_KO_Amice (FIG. 2C). Plasma phenylalanine levels were observed every week for11 weeks, e.g, as described in Gregory et al, Blood phenylalaninemonitoring for dietary compliance among patients with phenylketonuria:comparison of methods, Genetics in Medicine (November 2007) 9, 761-765,which is incorporated herein by reference. Two of three female micereceived 1×10¹² GC/kg of the vectors and all seven male ones with bothdoses at 1×10¹² GC/kg and 1×10¹³ GC/kg displayed a reduced phenylalanineconcentration in the plasma. The phenylalanine of the seven male micemaintained at comparably low levels for the 11-week observation periodwhile all three female ones demonstrated a slow increase in plasmaphenylalanine level.

Three female PAH_KO_B mice were generated and exanimated in a naturalhistory study. Weekly bleeds for phenylalanine levels were performed andthe result confirmed an abnormal accumulation of phenylalanine in theblood compared to the healthy littermate controls. Upon intravenousinjection of 1×10¹² GC/kg of AAV8.TBG.PI.hPAHco.WPRE.bGH, a decreasedphenylalanine level was observed in all three females and the low levelmaintained during the 8-week observation period after the injection.

Two male PAH_KO_C mice were generated for this study and utilized innatural history study. A weekly collection of blood samples wereperformed and the phenylalanine concentration was assessed. The datashowed that during the 9-week observation, both PAH_KO_C mice and theheterozygous/wild-type littermates maintained a comparably stableconcentration of plasma phenylalanine while the knock-out micedemonstrated a significantly higher level.

A further study of expression and enzyme activity of PAH in the injectedPAH^(−/−) mice was performed. Livers are collected from PAH_KO_A,PAH_KO_B and PAH_KO_C mice injected with vectors or PBS only as well asthe healthy littermate controls. mRNA is extracted from the liver andthe expression of human PAH is evaluated via RT-PCR. To determine theprotein expression of PAH, liver lysates are prepared for detection bywestern blot while liver sections are prepared for immunohistochemistry.Experiments are also performed to assess the PAH enzyme activity of thePAH^(-/-) mice treated with the vector as well as controls.

To fully evaluate gender difference in all three PAH^(−/−) mice,PAH_KO_A, PAH_KO_B and PAH_KO_C mice were bred to assess the plasmaphenylalanine concentration, the expression of PAH on both mRNA andprotein level and the enzyme activity of PAH before and after theinjection of AAV8.TBG.PI.hPAHco.WPRE.bGH.

To determine the dose-dependent expression ofAAV8.TBG.PI.hPAHco.WPRE.bGH and the potential toxicity of the highestdose, various doses of AAV8.TBG.PI.hPAHco.WPRE.bGH are injected into thePAH^(−/−) mice and further assessment of phenylalanine accumulation andPAH expression/activity are performed.

Similar experiments were performed with AAV8.TBG.hPAHco.bGH. PKU B miceages 17-22 weeks were given either 10¹² GC/kg of eitherAAV8.TBG.hPAHco.bGH or AAV8.TBG.hPAHco.WPRE.bGH (or PBS for control)after pretreatment phenylalanine levels were established. Mice were thenbled weekly, and plasma was isolated and phenylalanine concentration wasdetermined (FIG. 6A). At the termination of the study, liver wascollected and genome copy analysis (FIG. 6B) and immunohistochemistry(FIG. 6C) was performed. Phenylalanine levels were reduced in bothAAV8.TBG.hPAHco.bGH and AAV8.TBG.hPAHco.WPRE.bGH treated mice.

Further studies were performed with AAV8.TBG.hPAHco.bGH vector.Wildtype, heterozygous (circle) and PKU_KO_A (KO) mice were injectedintravenously with 10¹¹ GC/kg of AAV8.TBG.hPAHco after baselinephenylalanine levels were established. Mice were then bled weekly, andplasma was isolated and analyzed for Phe concentration. Phe levels inplasma decreased by 71% following intravenous administration ofAAV8.TBG.hPAHco. FIG. 7.

PKU_KO_B mice were given either 10¹² GC/kg, 3×10¹¹ GC/kg, or 10¹¹ GC/kg,of AAV8.TBG.hPAHco after baseline phenylalanine levels were established.Mice were then bled weekly, and plasma was isolated and analyzed for Pheconcentration. At termination of the study, liver was collected andgenome copy analysis and immunohistochemistry was performed. Proteinexpression and reduction in Phe levels were seen at a dose of 10¹²GC/kg.

Meanwhile, administration of 10¹² GC/kg of each of the followingvectors, AAV8.TBG.PI.hPAHco.bGH, AAV8.LSP.IVS2.hPAHco.bGH,AAV8.A1AT.hPAHco.BGH, AAV8.TTR.hPAHco.BGH,AAV8.TBG.PI.hPAHnativesequence.bGH, AAV8.ABPS.TBG.hFIXintron.hPAHco.BGH,AAV8.ABPS.TBG-S1.hFIXintron.hPAHco.BGH,AAV8.ApoE.A1AT.hFIXintron.hPAHco.BGH, is also performed and served as acomparison.

In conclusion, a single injection of AAV8.TBG.PI.hPAHco.WPRE.bGH vectorresulted in substantial plasma phenylalanine reduction and concomitantfunctional correction when administered intravenously in threePAH-deficient mice.

EXAMPLE 4: AAV Gene Therapy for Phenylketonuria

Phenylketonuria (PKU) is an autosomal recessive genetic disorder causedby the attenuation of phenylalanine-4-hydroxylase (PAH) activity,resulting in the buildup of phenylalanine in the tissues and blood. Highlevels of phenylalanine in the bloodstream are thought to inhibit thetransport of other large neutral amino acids across the blood brainbarrier, affecting brain development and resulting in intellectualdisability and seizures. Treatment for PKU is currently limited tomaintenance of a strict phenylalanine-restricted diet and productsdirected at stabilizing residual PAH. A liver-targeted AAV gene therapyapproach described herein is to improve upon the current standard ofcare.

To investigate the development of gene therapy for PKU, four uniquemouse strains were created by inducing different mutations in exon 1 ofthe PAH gene by CRISPR/Cas9 technology as described herein. A naturalhistory study was perforemed on each of these strains to determine theprogression of the disease and identify the strain that best replicatedthe human PKU phenotype. PKU colonies, designated B and C, bothcontained a single base pair (bp) deletion at different locations inexon 1 and maintained average phenylalanine levels of 2049 μM and 1705μM, respectively, compared to normal levels of 70 μM. PKU colony A,despite having a 64 bp deletion and a 3 bp insertion in exon 1 of thePAH gene, had a modestly higher average phenylalanine level of 477 μM.PKU colony D, which had a 6 bp deletion, had phenylalanine levelsequivalent to wild type littermates. Following AAV8 vectoradministration at a dose of 1×10¹² GC/kg for expression of a human codonoptimized version of PAH to the PKU B mouse colony, plasma phenylalaninelevels were reduced by 87% to 222 μM. This reduction in plasmaphenylalanine levels restored the ability of the males to produceoffspring. These results represent development of an AAV-basedtherapeutic for PKU.

All publications cited in this specification are incorporated herein byreference. Similarly, the SEQ ID NOs which are referenced herein andwhich appear in the appended Sequence Listing are incorporated byreference. While the invention has been described with reference toparticular embodiments, it will be appreciated that modifications can bemade without departing from the spirit of the invention. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A recombinant adeno-associated virus (rAAV) useful as aliver-directed therapeutic for phenylketonuria (PKU), said rAAVcomprising an AAV capsid, and a vector genome packaged therein, saidvector genome comprising: (a) an AAV 5′ inverted terminal repeat (ITR)sequence; (b) a promoter; (c) a codon optimized sequence encoding ahuman phenylalanine hydroxylase (PAH); (d) an AAV 3′ ITR.